U.S. patent application number 13/133384 was filed with the patent office on 2012-02-02 for method for amplifying oligonucleotide and small rna by using polymerase-endonuclease chain reaction.
This patent application is currently assigned to Xiaolong WANG. Invention is credited to Deming Gou, Cuixian I.V., Chenguang Liu, Xiaolong Wang.
Application Number | 20120028253 13/133384 |
Document ID | / |
Family ID | 42309765 |
Filed Date | 2012-02-02 |
United States Patent
Application |
20120028253 |
Kind Code |
A1 |
Wang; Xiaolong ; et
al. |
February 2, 2012 |
METHOD FOR AMPLIFYING OLIGONUCLEOTIDE AND SMALL RNA BY USING
POLYMERASE-ENDONUCLEASE CHAIN REACTION
Abstract
A method for amplifying oligonucleotide in vitro by
polymerase-endonuclease chain reaction (PECR) which utilizes a
single-stranded DNA probe containing repeat sequences, extends a
target oligonucleotide by a thermostable DNA polymerase, cleaves
extended products with a thermostable endonuclease, and amplifies
target oligonucleotide by thermocycling. In PECR, a specific
oligonucleotide is exponentially amplified using one single probe
instead of a pair of primers, and the reaction is precisely
controlled by thermal cycles whose parameters are flexibly
adjustable according to length, sequence, melting temperature and
initial amount of the target oligonucleotide. Amplification speed
depends totally on initial amount of target oligonucleotide present
in the reaction system. The method can be used to amplify specific
small nucleic acids, such as oligonucleotides and microRNAs, and
further conduct quantitative analysis. PECR is easy to conduct with
high efficiency, specificity and stability, and thus can be widely
used in molecular biology studies.
Inventors: |
Wang; Xiaolong; (Shandong,
CN) ; I.V.; Cuixian; (Shandong, CN) ; Gou;
Deming; (Shandong, CN) ; Liu; Chenguang;
(Shandong, CN) |
Assignee: |
WANG; Xiaolong
|
Family ID: |
42309765 |
Appl. No.: |
13/133384 |
Filed: |
April 3, 2009 |
PCT Filed: |
April 3, 2009 |
PCT NO: |
PCT/CN2009/000362 |
371 Date: |
September 2, 2011 |
Current U.S.
Class: |
435/6.11 ;
435/91.2; 435/91.21 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 1/6851 20130101; C12Q 1/6851 20130101; C12Q 2525/151 20130101;
C12Q 2561/113 20130101; C12Q 2525/207 20130101; C12Q 2525/151
20130101; C12Q 2525/131 20130101; C12Q 2525/207 20130101 |
Class at
Publication: |
435/6.11 ;
435/91.2; 435/91.21 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 5, 2009 |
CN |
200910300070.8 |
Claims
1. A polymerase-endonuclease chain reaction for the amplification
of oligonucleotides and small RNAs. The method comprising: 1) The
composition of the reaction mixture: (1) A target nucleic acid
sequence X, either double-stranded or single-stranded, length of 8
to 50 bases or base pairs, and its melting temperature (Tm) in the
range of 36.about.79.degree. C.; (2) An antisense probe, denoted by
X'R'X', is designed to be a single-stranded oligonucleotide
containing at least two tandem repeated complements of the target
sequence (X') that are separated from one another by an intervening
complementary recognition site (R') for a restriction endonuclease;
(3) A thermostable DNA polymerase; (4) A thermostable restriction
endonuclease; (5) Four deoxyribose nucleotides triphosphate: dATP,
dGTP, dCTP and dTTP; (6) An appropriate buffer solution; 2) The
thermocycling reaction: the above reaction mixture is incubated at
60.quadrature. to 99.quadrature. for 0.about.600 seconds of
pre-denaturation, then subject to 1-100 cycles of thermocycling,
each thermal cycle consists the following four steps: (1)
Denaturing: incubate the reaction mixture in a temperature at least
5.quadrature. above the melting temperature of the target nucleic
acid. The temperature ranges from 60.about.990, duration ranges
from 1 to 60 seconds; (2) Annealing: incubate the reaction mixture
in a temperature equal to, or within 5.quadrature. higher or lower
than, the melting temperature of the target nucleic acid. The
temperature ranges from 35.about.68.quadrature., duration ranges
from 1 to 60 seconds; (3) Elongation: incubate the reaction mixture
in a temperature at least 5.quadrature. above the melting
temperature of the target nucleic acid, and within the optimal
working temperature of the said DNA polymerase. The temperature
ranges from 45 to 89.quadrature., duration ranges from 1 to 60
seconds; (4) Cleaving: Insulation of the reaction mixture in a
temperature at least 5.quadrature. above the melting temperature of
the target nucleic acid, and within the optimal operating
temperature of the restriction enzymes the said. The temperature
ranges from 45.about.89.quadrature., duration ranges from
1.about.300 seconds; the temperatures of (1) denaturing, (3)
elongation and (4) cleaving steps are at least 10.quadrature.
higher than the annealing temperature in step (2). By repeated
steps (1) to (4), say denaturation, annealing, extension and
cleaving, the target nucleic acid molecules are amplified
exponentially, the products include double-stranded repetitive
nucleic acid XRX/X'R'X', double-stranded target nucleic acid X/X'
and single-stranded target molecule X.
2. The method of claim 1 wherein the said DNA polymerase is a hot
start thermostable DNA polymerase. The said restriction enzyme is a
thermostable double-stranded endonuclease.
3. The method of claim 1 wherein the said target nucleic acid is
any synthetic or natural DNA molecules, including oligonucleotides,
genomic DNA, mitochondrial DNA, cDNA derived from reverse
transcription of mRNA, microRNA, or siRNA.
4. The method of claim 1 wherein the said target nucleic acid is
RNA molecules, including mRNA, microRNA and siRNA, or any other
kind of synthetic or natural RNA molecules, and a DNA polymerase
that can elongate RNA molecules directly is included in the said
reaction mixture.
5. The method of claim 1 wherein the said antisense probe contains
two or more tandem repeats of the complementary sequence (A') of
the target sequence (A). Between two adjacent repeats, there is at
least one recognition sites (R') of a thermostable endonuclease.
The general molecular formula of the probe is A'-(R'A').sub.n,
where n is a positive integer greater than or equal to 1.
6. The method of claim 1 wherein the said antisense probe contains
two or more different complementary target sequences (A', B', C').
Between two adjacent target sequences, there is at least one
recognition sites (R') of a thermostable endonuclease. The
molecular formulas of the probe are A'-(R'B').sub.n, B'R'A'--(R'
B').sub.n, or A'R'B'--(R'C').sub.n, where n is a positive integer
greater than or equal to 1.
7. The method of claim 1 wherein the end or the middle of the said
probe contains an isotope labeled nucleotides.
8. The method of claim 1 wherein the said the reaction mixture
contains a DNA-specific binding fluorescent dye.
9. The method of claim 1 wherein the end or the middle of the said
probe in connection with one or more chemical groups.
10. The method of claim 9 characterized in that one of the said
chemical groups is fluorophores.
11. The method of claim 1 wherein the end or the middle of the of
the said target nucleic acid containing fluorophores.
12. The method of claim 1 wherein the said probe is methylated.
13. The method of claim 1 wherein the said probe is fixed on a gene
chip or other solid surface.
14. The method of claim 1 wherein the end of the said probe is
connected to a nano-material.
Description
BACKGROUND OF THE PRESENT INVENTION
[0001] 1. Field of Invention
[0002] The present invention relates to the field of molecular
biology and gene engineering. More specifically, the present
invention is a method for amplifying oligonucleotides and small RNA
molecules.
[0003] 2. Description of Related Arts
[0004] Nucleic acid amplification technology is at the core of
contemporary molecular biology and gene engineering. In recent
years, with the emerging of novel nucleic acid amplification
methods, many new nucleic acid amplification based detection and
diagnosis approaches have been developed and widely used. Although
these methods have encountered some problems in practice, such as
false positives and false negatives, they have many advantages,
especially for small amount of sample requirement, rapid, sensitive
and accurate. Therefore, many researchers from worldwide dedicated
in developing new nucleic acid amplification methods, or improving
existing technologies.
[0005] According to whether the reaction temperature changes or
not, nucleic acid amplification methods can be divided into two
broad categories: respectively thermocycling amplification methods
and isothermal amplification methods. Classical thermocycling
amplification methods include polymerase chain reaction (PCR) and
ligase chain reaction (LCR); recent emerging isothermal
amplification methods mainly include strand displacement
amplification (SDA), rolling circle amplification (RCA),
loop-mediated amplification (LAMP), helicase-dependent isothermal
amplification (HDA), nucleic acid sequence based amplification
(NASBA), and transcription-based amplification system (TAS), and so
on.
[0006] Making a comprehensive survey at current nucleic acid
amplification technologies in molecular biology, molecular
diagnosis and gene engineering, in spite of many novel nucleic acid
amplification approaches has been reported, PCR (U.S. Pat. Nos.
4,683,195 and 4,683,202) is still the most commonly used method for
in vitro nucleic acid amplification. PCR and reverse transcription
PCR (RT-PCR) is simple and effective for amplification of DNA and
mRNA with sufficient length. But it is not able to be used directly
for amplifying small nucleic acids, such as oligonucleotides,
microRNAs (miRNAs) and small interfering RNAs (siRNAs). The reverse
transcript of a miRNA is actually an oligonucleotide that is
generally only 18-25 nucleotides in length. Therefore, the length
of miRNA-derived cDNA is usually insufficient for designing a pair
of specific primers, which is a prerequisite of PCR.
[0007] Oligonucleotides have a wide range of applications in modern
molecular biology studies. Although a large quantity of a synthetic
oligonucleotide that have an identical sequence can be accurately
quantified by spectrophotometry analysis according to its optical
density (OD), amplification and quantification of trace amount of
target oligonucleotide in a biological sample that contains a lot
of different nucleic acid sequences, is still an unsolved technical
problem, which may have important applications in science. For
example, it can be used for preparation of oligonucleotides and
quantitative analysis of small RNAs such as miRNA and siRNA.
[0008] However, only a few studies on the amplification of
oligonucleotides have been reported. The international patent
application, PCT/US04/02718, entitled "Isothermal Reactions for the
Amplification of Oligonucleotides", described the Exponential
Amplification Reaction (EXPAR) that is capable of amplifying
oligonucleotides. The method relies on polymerase, nicking enzymes
and strand displacement, using a repeat-containing single-stranded
DNA (ssDNA) as template, 10.sup.7-fold amplification of the target
oligonucleotide can be achieved in 5 min. The reaction is very
easy, fast, and carried out at constant temperature conditions,
without the need of a PCR instrument. However, in a recent study it
is pointed out that serious non-specific background amplification
and false positives exist in the reaction (Tan E, et al, Specific
versus nonspecific isothermal DNA amplification through
thermophilic polymerase and nicking enzyme activities. Biochemistry
2008, 47 (38): 9987-9999). Hence, the method is unreliable for use
in quantitative analysis of nucleic acids.
[0009] On the other hand, microRNA, or miRNA in short, is a class
of endogenous small regulatory RNA, about 20-24 nucleotides in
length. In 1999, miRNA lin-4 was firstly found in nematodes. Since
then, a lot of miRNAs with important roles in gene regulation were
found in worms, fruit flies, mice, zebra fish and other model
organisms and humans. MiRNA binds to its target, protein coding
messenger RNA (mRNA), in the 3'-untranslated region (3'-UTR) with
full complementarility inducing target mRNA degradation, or with
partial complementarility inhibiting translation of the target
mRNA, which is called post-transcriptional gene silencing (PTGS).
MiRNAs involve in the regulation processes, and play important
roles in the basic life activities in a lot of organisms. For
example, lin-4 involved in controlling nematodes timing of larval
development, mir-14 controls Drosophila cell death and fat
metabolism. In Zebrafish, miR-214 determines the fate of muscle
cells, and miR-430 functions the removal of maternal mRNAs that are
no longer needed in early embryos. MiR-375 is a highly conserved
islet-specific miRNA family. In Zebrafish, miR-375 determines islet
development. Reduced levels of miR-375 inhibited the aggregation of
islet cells and insulin secretion in humans. The role of miR-375 in
other model organisms and humans is highly conserved, suggesting
that the function of miR-375 is conservative from Zebrafish to
human. The functional importance of miRNAs has attracted
researchers worldwide to study the origin, mechanism and function
of miRNA using a variety of model organisms.
[0010] As of Mar. 6, 2009, the miRNA database (miRbase Release
12.0) has a collection of 8619 miRNA sequences. However, in
contrast to the rapid discovery of new miRNAs, progress in miRNA
functional studies is far slower. By far there are only a few
miRNAs whose function is well characterized. For functional miRNA
research, first is to determine the target genes of the miRNA and
its regulatory function, second is to study the temporal and
spatial expression of its own regulation by quantitative detection.
Since the timing and the tissue-specificity of miRNA expression is
useful in revealing their function in a specific tissue and cell.
The main cause of the slow progress in miRNA functional studies has
two points: firstly, it is difficult to determine their target
genes; secondly, the amplification and quantification of small RNAs
is far more difficult than that of long messenger RNAs.
[0011] At present, amplification and detection of miRNA are based
on real time quantitative reverse transcription-PCR (qRT-PCR).
Because mature miRNAs have no poly (A) tails, reverse
transcription-PCR methods for miRNA are different from that for
mRNA. There are mainly two strategies: first, miRNAs is
polyadenalated using Poly (A) Polymerase (PAP) to add poly (A)
tails to the 3'-end, and then synthesis cDNA by using Oligo (dT)
and reverse transcriptase. Another method is using a miRNA-specific
primer (miSP) for reverse transcription. The miSP contains a
specific sequence complementary to the 3'-end of the miRNA and a
stem loop structure. Since miRNA is too short to design a pair of
specific primers, therefore, no matter which method is used,
reverse transcription must be conducted using a downstream primer
containing a universal tag, by which the universal tag is
introduced into the cDNA. And then the resulting cDNA is amplified
by PCR using a miRNA-specific primer as upstream primer and a
universal primer as downstream primer.
[0012] The efficiency of polyadenalation and reverse transcription
is of crucial importance to the accuracy of quantitative analysis.
However, using an extra-long primer with a universal tag for
reverse transcription will cause a decline in the efficiency of
reverse transcription, and thus have a negative impact on the
accuracy of the quantification of the miRNA. In addition, in PCR,
the difference of the melting temperatures (Tm) between the
upstream and the downstream primers should not exceed more than
2.degree. C. However, for many miRNA-specific primers, their Tm
inevitable differ more than 5.degree. C. from the universal primer,
because a same universal primer is not fit for all miRNA sequences.
Some miRNA-specific primers and the universal primer will bind to
each other to form primer dimers, resulting in non-specific
amplification, false positive and false negative problems.
SUMMARY OF THE PRESENT INVENTION
[0013] Current available nucleic acid amplification methods, such
as PCR, have difficulties in amplification of oligonucleotides and
small RNAs. This invention presents a new nucleic acid
amplification method, designated as polymerase-endonuclease chain
reaction (PECR), or polymerase-endonuclease amplification reaction
(PEAR). PECR uses only one single ssDNA probe to amplify a specific
miRNA or a target oligonucleotide. The PECR method comprises
utilizing a repeat-containing ssDNA probe, repeatedly extending the
target oligonucleotides by a thermostable DNA polymerase, and
cleaving the extended products with a highly thermostable
restriction endonuclease. PECR is able to amplify a specific
oligonucleotide exponentially by only one probe instead of a pair
of primers that is generally required in traditional nucleic acid
amplification methods. The process of PECR is controlled by
thermocycling. The parameters of the thermal cycles are flexibly
adjustable according to the length, sequence, melting temperature
and initial concentration of the target oligonucleotide. The
reaction rate depends totally on the initial concentration of the
target oligonucleotide in the reaction mixture. The method can
amplify and quantify a specific small nucleotide acid, such as
oligonucleotide or microRNA, from a small biology sample. PECR
amplification of target oligonucleotides is conducted by
thermocycling and without using a universal primer, it is thus
simple, stable, efficient, and with high specificity, and thus can
be widely useful in molecular biology studies.
[0014] The present invention is implemented by the following
protocol: a method of polymerase-endonuclease chain reaction for
the amplification of oligonucleotides and small RNAs, the method
comprises:
[0015] 1) The composition of the reaction mixture:
[0016] (1) A target nucleic acid sequence X, either double-stranded
or single-stranded, length of 8 to 50 bases or base pairs, and its
melting temperature (Tm) in the range of 36.about.79.degree.
C.;
[0017] (2) An antisense probe, denoted by X'R'X', is designed to be
a single-stranded oligonucleotide containing at least two tandem
repeated complements of the target sequence (X') that are separated
from one another by an intervening complementary recognition site
(R') for a restriction endonuclease;
[0018] (3) A thermostable DNA polymerase;
[0019] (4) A thermostable restriction endonuclease;
[0020] (5) Four deoxyribose nucleotides triphosphate: dATP, dGTP,
dCTP and dTTP;
[0021] (6) An appropriate buffer solution;
[0022] 2) The thermocycling reaction: the above reaction mixture is
incubated at 60.degree. C. to 99.degree. C. for 0.about.600 seconds
of pre-denaturation, then subject to 1-100 cycles of thermocycling,
each thermal cycle consists the following four steps:
[0023] (1) Denaturing: incubate the reaction mixture in a
temperature at least 5.degree. C. above the melting temperature of
the target nucleic acid. The temperature ranges from
60.about.99.degree. C., duration ranges from 1 to 60 seconds;
[0024] (2) Annealing: incubate the reaction mixture in a
temperature equal to, or within 5.degree. C. higher or lower than,
the melting temperature of the target nucleic acid. The temperature
ranges from 35.about.68.degree. C., duration ranges from 1 to 60
seconds;
[0025] (3) Elongation: incubate the reaction mixture in a
temperature at least 5.degree. C. above the melting temperature of
the target nucleic acid, and within the optimal working temperature
of the said DNA polymerase. The temperature ranges from 45 to
89.degree. C., duration ranges from 1 to 60 seconds;
[0026] (4) Cleaving: Insulation of the reaction mixture in a
temperature at least 5.degree. C. above the melting temperature of
the target nucleic acid, and within the optimal operating
temperature of the restriction enzymes the said. The temperature
ranges from 45.about.89.degree. C., duration ranges from
1.about.300 seconds;
[0027] The temperatures of (1) denaturing, (3) elongation and (4)
cleaving steps are at least 10.degree. C. higher than the annealing
temperature in step (2). By repeated steps (1) to (4), say
denaturation, annealing, extension and cleaving, the target nucleic
acid molecules are amplified exponentially, the products include
double-stranded repetitive nucleic acid XRX/X'R'X', double-stranded
target nucleic acid X/X' and single-stranded target molecule X.
[0028] In this invention, the said thermostable DNA polymerase
could resist high temperatures above 80.degree. C. The best suited
DNA polymerase is a hot start thermostable DNA polymerase. The said
thermostable endonuclease is a double-stranded restriction enzyme
which could resist high temperatures above 80.degree. C.
[0029] The said target nucleic acid could be any kind of natural or
synthetic DNA molecule, including oligonucleotides, genomic DNA,
mitochondrial DNA, cDNA derived from reverse transcription of mRNA,
microRNA, or siRNA, and so on. The said target nucleic acid could
also be any type of synthetic or natural RNA molecules, including
mRNA, microRNA and siRNA, and so on. If a DNA polymerase that can
directly elongate RNA molecule, such as E. coli DNA polymerase I,
is included in the reaction mixture, PECR reaction can also be used
for direct amplification of RNA, particularly small RNAs, such as
siRNA and miRNA.
[0030] The antisense probe may contain two or more tandem repeats
of the complementary sequence (A') of the target sequence (A).
Between two adjacent repeats, there is at least one recognition
sites (R') of a thermostable endonuclease. The general molecular
formula of the probe is A'-(R'A').sub.n, where n is a positive
integer greater than or equal to 1. Such kind of probe with
multiple repeats enables faster rate per cycle of
amplification.
[0031] The antisense probe may contain two or more different
complementary target sequences (A', B', C'). Between two adjacent
target sequences, there is at least one recognition sites (R') of a
thermostable endonuclease. The molecular formulas of the probe are
A'-(R'B').sub.n, B'R'A'-(R' or A'R'B'--(R'C').sub.n, where n is a
positive integer greater than or equal to 1. Using such kind of
probe containing different target sequences, different target
sequences can be produced in a single PECR reaction. Moreover, one
or more oligonucleotide inputs can be used to output another one or
more target sequence(s), which can be useful in DNA circuit or DNA
computing.
[0032] The end or the middle of the said antisense probe may
contain one or more isotope labeled nucleotides, the labeled
nucleotides can be introduced into the amplification product in
random or predefined locations, so that the PECR product can be
detected using radioactive detection methods.
[0033] The said reaction mixture may contain a DNA-specific
fluorescent dye, including but not limited to Sybr Green I and Sybr
Green II, so that the fluorescence intensity of the reaction
mixture enhances with each round of PECR amplification, and the
fluorescence signal can be detected by real-time fluorescence
quantitative PCR instruments, and thus the initial amount of the
target oligonucleotide can be quantitatively measured.
[0034] The middle or the end of the probe can be connected to one
or more chemical groups, including but not limited to,
fluorophores, quenching group, biotin, digoxin, amino acids, amino,
amino-C3, amino-C6, amino-C12, amino-C18, Tsuen base, carboxyl,
sugar ring, peptides, peptide nucleic acid, and so on.
[0035] The end or the middle of the said probe may contain a
fluorophore and quencher groups, the fluorophore are located on
one, and the quencher is on another, side of the restriction site.
When the restriction sites were cleaved in a PECR reaction that
makes the fluorophore and quencher separate from each other, the
fluorescence intensity of the reaction mixture increased, which can
be monitored by a real-time quantitative PCR instrument, so that
the initial copy number of the target oligonucleotide can be
quantitatively analyzed.
[0036] The end or the middle of the said target oligonucleotide may
contain a fluorophore and a quencher group. When the restriction
sites were cleaved in a PECR reaction that makes the fluorophore
and quencher separate from each other, the fluorescence intensity
of the reaction mixture increased, which can be monitored by a
real-time quantitative PCR instrument, so that the initial copy
number of the target oligonucleotide can be quantitatively
analyzed.
[0037] The restriction sites in the said probe could be methylated,
so that the restriction site could not be cut by endonuclease, but
it could be cut after being demethylated. Note that in the PECR
products the restriction sites could usually be cut because they
are not methylated.
[0038] The said probe can be fixed in a gene chip, or the surface
of solid materials, particles or plates, so that a large number of
different target oligonucleotides could be detected by a high
throughput method. The matrix could be made by various materials
including silicon materials such as silicon or silicon dioxide
film, silicon substrate, silicon nanowires, conductive metals such
as gold, platinum, carbon materials, such as graphite, carbon
nanotubes, and conductive resin, and so on. Some materials can also
be used in the form of particles or beads, in which the probe is
connected to the surface of these materials, so that PECR reactions
could occur on the surface of them.
[0039] The end of the said probe could be connected with
nano-materials, so nano-materials can be used to detect PECR
reaction, or use PECR reactions to control nano-materials.
Nano-materials are kinds of materials with zero-dimensional,
one-dimensional, two-dimensional, or three-dimensional structures,
which are composed of ultra-fine structures with small size effects
(the size are smaller than 100 nm, ranges 0.1-100 nm). The shapes
of nano-materials include nanowires, nanorods, nanotubes,
nanobelts, nano-particles, nano-film, nano-crystals, nano
non-crystalline, nano-fibers, nano-bulk, etc. Nano-materials
include but not limited to, carbon nanotubes, nano-fullerenes (such
as carbon sixty), nano-ceramics, nano-metal particles, zinc oxide
particles, nano silica, nano-titanium dioxide and iron oxide
nanoparticles. Nano-materials also include bio-nano-materials,
which are biological macromolecules, such as polypeptide chains,
polysaccharides, amino-polysaccharides and nucleic acids, and so
on.
[0040] PECR product can be detected by the polyacrylamide gel
electrophoresis (PAGE). Preparing a non-denaturing polyacrylamide
gel with a concentration of 12%.about.15%, and 5.about.15 cm in
length, running through a 250.about.300V electrophoresis for
20.about.40 min, the DNA bands can be visualized by one of the
following methods:
[0041] (1) Staining the gel with ethidium bromide dye, then observe
and photograph the DNA bands with UV gel imaging system;
[0042] (2) Staining the gel with Sybr Green I or Sybr Green II dye,
and then observe and photograph the DNA bands with UV gel imaging
system;
[0043] (3) Reveal DNA bands by silver staining;
[0044] (4) Mix radioisotope labeled single deoxyribonucleotide into
the PECR reaction system, then operate electrophoresis followed by
autoradiography imaging.
[0045] Real-time fluorescence quantitative detection can also be
performed on PECR product with the following two methods:
[0046] (1) Adding fluorescent dye directly into the reaction
mixture:
[0047] Adding Sybr Green I or II fluorescent dye into the reaction,
Sybr Green binds specifically with the minor groove of DNA with
high affinity for double-stranded DNA (dsDNA), while its binding
capacity with single-stranded DNA (ssDNA) is very low. At the
beginning of PECR reaction, the probe is single-stranded, thus
binds with the Sybr Green weakly, and the fluorescence intensity is
at a relatively low level. During PECR reaction cycles,
single-stranded probe were converted into double-stranded products.
The fluorescence intensity enhance greatly due to Sybr Green dyes
bind with double-stranded products, which can be detected with a
fluorescence quantitative real-time PCR instrument, such as ABI
7500.
[0048] (2) Labeling PECR probe with fluorophore and quencher
groups:
[0049] Since Sybr Green dyes binds with dsDNA nonspecifically,
quantification of nucleic acids based on them have the
false-positive problem: if a false-positive or a nonspecific
amplification occurred, it is not distinguishable from a true
positive reaction. For the purpose of more accurate quantitative
detection, one can label the PECR probe with fluorophore and
quencher.
[0050] Fluorophores that can be used include but are not limited
to: 6-carboxyfluorescein (FAM), Tetrachlorofluorescein (TET),
hexachlorofluorescein (HEX),
N,N,N,N'-tetramethyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine(ROX),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), fluorescein
isothiocyanate (FITC),
3-(-carboxy-pentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine (CyA);
Texas Red, 6-carboxyrhodamine (R6G) etc. Quenchers include but not
limited to TARMA, Iowa Black (IWB), etc.
[0051] The said target nucleic acid of PECR reaction can be any DNA
molecules, including oligonucleotides, genomic DNA, mitochondrial
DNA, cDNA reverse transcript from mRNA, microRNA, or siRNA, and any
other DNA molecules. PECR reaction can also be used for amplifying
RNA directly, particularly siRNA, miRNA or other small RNA
molecules. For different target nucleic acids, the technical
protocols used are as followed:
[0052] (1) For single-stranded or double-stranded oligonucleotides
which has a length of 8 to 50 base pairs, use the basic PECR
protocol to do amplification;
[0053] (2) For microRNA or siRNA, use the reverse transcript PECR
(RT-PECR) protocol, which first reverse transcript the microRNA or
siRNA into cDNA, and then conduct the amplification by PEAR, or
adopt the RNA direct PECR (RD-PECR) protocol to amplify the target
RNA directly;
[0054] (3) For a long nucleic acid which has more than 50 base
pairs, this method can only amplify the specific sequence with the
length about 8 to 50 base pairs in the 3'-end, but not the full
sequence;
[0055] (4) For a specific sequence in the middle of a long target,
firstly use an endonuclease, whose recognition site is closely
adjacent to the target sequence, to cut the target DNA, letting the
target sequence exposed to the 3'-end, and then amplify by
PECR.
[0056] The present invention is the first to disclose the
polymerase-endonuclease chain reaction (PECR), or
polymerase-endonuclease amplification reaction (PEAR), which is a
new nucleic acid amplification technology. The difference between
PEAR and other nucleic acid amplification technology are as
followed:
[0057] (1) The comparison of PECR and other existing DNA
Amplification technologies: PCR amplify linear or circular DNA to
single copy linear fragments through thermal cycling; RCA amplify
circular DNA to linear multi-copy tandem repeat DNA molecules
through isothermal reaction; LAMP amplify linear DNA to linear
multi-copy tandem repeat DNA through isothermal reaction; EXPAR use
tandem repeat DNA template to amplify oligonucleotides through
isothermal reaction; PECR use tandem repeat DNA probe to amplify
small nucleic acids through a thermal cyclic reaction, so the PECR
technology presented in this invention is an important new member
of the family of nucleic acid amplification technologies.
[0058] (2) The comparison between PECR and PCR technique: the
principle of PECR is different from that of PCR, The major
differences include: (1) PCR depends only on thermostable DNA
polymerase, while PECR depends not only on thermostable DNA
polymerase, but also on thermostable endonuclease; (2) PCR requires
at least a pair of primers, but PECR needs only one single
repeat-containing probe; (3) In a PCR reaction, primers are only
extended, while in a PECR the repeat-containing products is not
only extended but cleaved, so that the copy number of product
molecules increases in each cycle; (4) PCR is not able to directly
amplify nucleic acid whose length is too short, while PECR is
designed to directly amplify nucleic acid of a shorter length,
particularly oligonucleotides and small RNAs; (5) PCR products are
usually longer than primers used, while PECR products are shorter
than the probe; (6) In each cycle of PCR amplification, products
can only be doubled at most, and the total number of final product
molecules could not exceed the number of input primers, while in
PECR using probes with more tandem repeats, products can be
increased more than two-fold for every cycle, and the copy number
of final product molecules far exceeds the copy number of input
probes;
[0059] (3) The comparison between PECR and coupled PCR-restriction
endonuclease digestion (PCR-RED): in patent PCT/US2000/007133
titled "COUPLED POLYMERASE CHAIN REACTION-RESTRICTION ENDONUCLEASE
DIGESTION-LIGASE DETECTION REACTION PROCESS", which described
PCR-restriction endonuclease digestion, which can eliminate or
significantly reduce the formation of non-specific PCR products.
Although the reaction both adopt the DNA polymerase and
thermostable restriction enzymes, the principles of PECR and
PCR-RED are fundamentally different: the basic principle of the
PCR-RED is still same to PCR and the thermostable restriction
enzymes play only an assistant role. The purpose is to eliminate or
reduce the non-target DNA amplification which contains restriction
sites of thermostable restriction enzymes by enzyme digestion, but
not to achieve exponentially amplification of the target DNA. While
in PECR, the role of thermostable restriction enzyme is not to
eliminate non-target DNA amplification, but the key enzymes to
achieve exponentially amplification of the target DNA.
[0060] (4) The comparison between PECR methods and EXPAR Methods:
In the patent "Isothermal reactions for the Amplification of
oligonucleotides" (PCT/US04/02718) described an isothermal
exponential amplification reaction that is called EXPAR reaction.
Both the present invention PECR and EXPAR reaction use the same
probe design strategy, but PECR reaction and EXPAR reaction are
fundamentally different: (1) EXPAR is an isothermal amplification
reaction, the process of EXPAR reaction is not controlled, while
PECR reaction process is tightly controlled by thermal cycling; (2)
EXPAR depends on a single strand nicking enzyme, while PECR adopts
a double-stranded endonuclease; (3) EXPAR is not able to use a
hot-start DNA polymerase, so that only manually hot start can be
applied, while PECR can be automatically hot started by a
thermalcycler using hot-start DNA polymerase; (4) EXPAR reaction
has seriously non-specific background amplification and
false-positive problem, while PECR reaction has little non-specific
background amplification and can in principle overcome the false
positive problem thanks to the tightly controlled thermal
cycling.
[0061] Although Tan et al. reported a EXPAR reaction which was
carried out by manually hot start to reduce non-specific
amplification, but hot-starting of the EXPAR, do not like PCR,
could not be implemented automatically by a PCR thermalcycler using
hot-start DNA polymerase (Tan E, et al, Specific versus nonspecific
isothermal DNA amplification through thermophilic polymerase and
nicking enzyme activities. Biochemistry. 2008, 47 (38): 9987-9999).
A hot start polymerase, which is a reversible inactivation of a DNA
polymerase prepared through chemical modification or
anti-polymerase antibody, could not be used in EXPAR. Because the
hot start polymerase must be heat activated at above 90.degree. C.
for about 10 min, while EXPAR reaction depends on a strand
displacement activity of the Bst polymerase and the nicking enzyme
Nb. BstNBI, which would be both heat inactivated in such a high
temperature. Although there are some strand displacement DNA
polymerases (such as VentR exo-) that could resist high temperature
above 90.degree. C., but at present highly thermostable nicking
enzyme, which can resist high temperatures above 90.degree. C., is
not available. Therefore, the EXPAR reaction must be hot start
manually: first heat reaction mixture to a predetermined
temperature, then add the DNA polymerase and the nicking enzyme.
Manual hot start is not only cumbersome, but could not be
implemented for real time quantitative analysis, greatly limited
the application of the method.
[0062] In contrast, PECR reactions can be conducted using a hot
start DNA polymerase, so that automatic hot started by a PCR
instrument with the inherent reliability and convenience. In
addition, as in the PCR reaction, the PECR process is tightly
controlled by thermal cycling, and the reaction parameters
including annealing temperature, annealing time, number of cycles,
and so on, is flexibly adjustable according to the length,
sequence, melting temperature, and the initial number of the target
oligonucleotides.
[0063] In summary, PECR is a new, simple but effective nucleic acid
amplification technology. By PECR, using a specific probe, we can
selectively amplify a specific small nucleic acid, including
oligonucleotides and miRNAs, with a known sequence quantitatively,
rapidly, accurately and sensitively. PECR is easy to adapt for
fully automation and real-time quantitative detection, thus could
be widely useful in molecular biology studies, e.g. amplification
and quantification of miRNAs and small RNAs for gene expression
profiling, gene chip technologies, high-throughput nucleic acid
detection, large-scale amplification or preparation of antisense
oligonucleotides, intelligent nucleic acid detection and molecular
computing, etc.
[0064] Still further objects and advantages will become apparent
from a consideration of the ensuing description and drawings.
[0065] These and other objectives, features, and advantages of the
present invention will become apparent from the following detailed
description, the accompanying drawings, and the appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1. The schematic diagram of polymerase-endonuclease
chain reaction;
[0067] FIG. 2. The schematic diagram of fluorescent labeling of
PECR probe;
[0068] FIG. 3. Verification of PECR reaction principle (Embodiment
1);
[0069] FIG. 4. Electrophoresis result of amplification with
different initial concentration of target oligonucleotides
(Embodiment 1);
[0070] FIG. 5. The schematic diagram of reverse transcriptase PECR
(RT-PECR);
[0071] FIG. 6. The schematic diagram of RNA-direct PECR
(RD-PECR);
[0072] FIG. 7. A result of real-time fluorescence detection of PECR
products (Embodiment 6).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Examples of Embodiment
Embodiment 1
[0073] In this example of embodiment, polymerase-endonuclease chain
reaction is implemented to amplify oligonucleotides using
thermostable DNA polymerase and thermostable restriction
endonuclease that can cut double-stranded DNA. The said
thermostable DNA polymerase and thermostable endonuclease can
resist high temperature above 50.degree. C., and its optimum
working temperature range is 45-89.degree. C. The thermostable DNA
polymerase includes but not limits to Taq DNA polymerase, DyNAzyme
II DNA Polymerase.RTM., LA Taq DNA Polymerase.RTM., Pfu DNA
polymerase .RTM., VentR DNA Polymerase.RTM., Deep VentR DNA
Polymerase.RTM., VentR exo-DNA Polymerase.RTM., Deep VentR (exo-)
DNA Polymerase.RTM., 9.degree. Nm DNA Polymerase.RTM., etc. A
hot-start DNA polymerase will be better for use in this reaction.
Hot-start DNA polymerases include but not limits to hot-start Taq
DNA polymerase, DyNAzyme II Hot Start DNA Polymerase.RTM., KOD
Xtreme Hot Start DNA Polymerase.RTM., Phusion DNA Polymerase.RTM.,
Pfu Ultra type of hot start DNA Polymerase.RTM., Platinum DNA
polymerase and Thermo-Start DNA Polymerase.RTM., etc. Thermostable
restriction enzymes include but not limited to PspGI, ApeKI, BstUI,
BstNI, MwoI, Phol, TseI, Tsp451, Tsp5091, TspRI and TfiI, etc.
[0074] The method comprises the following:
[0075] 1) The composition of the reaction mixture:
[0076] (1) A target nucleic acid X, either double-stranded or
single-stranded, length is in the range of 8 to 50 bases or bp, and
its melting temperature is in the range of 36.about.79.degree.
C.;
[0077] (2) An antisense probe, denoted by X'R'X', is designed to be
a single-stranded oligonucleotide containing at least two tandem
repeats of complement target sequence (X') that are separated from
one another by an intervening complementary recognition site (R')
for a restriction endonuclease;
[0078] (3) A thermostable DNA polymerase;
[0079] (4) A thermostable restriction endonuclease;
[0080] (5) Four deoxyribose nucleotides triphosphate: dATP, dGTP,
dCTP and dTTP;
[0081] (6) An appropriate buffer solution;
[0082] 2) The thermocycling reaction: the above reaction mixture is
incubated at 60.degree. C. to 99.degree. C. for 0.about.600 seconds
of pre-denaturation, then subject to 1-100 cycles of thermocycling,
each thermal cycle consists the following four steps:
[0083] (1) Denaturing: incubate the reaction mixture in a
temperature at least 5.degree. C. above the melting temperature of
the target nucleic acid. The temperature ranges from
60.about.99.degree. C., duration ranges from 1 to 60 seconds;
[0084] (2) Annealing: incubate the reaction mixture in a
temperature equal to, or within 5.degree. C. higher or lower than,
the melting temperature of the target nucleic acid. The temperature
ranges from 35.about.68.degree. C., duration ranges from 1 to 60
seconds;
[0085] (3) Elongation: incubate the reaction mixture in a
temperature at least 5.degree. C. above the melting temperature of
the target nucleic acid, and within the optimal working temperature
of the said DNA polymerase. The temperature ranges from 45 to
89.degree. C., duration ranges from 1 to 60 seconds;
[0086] (4) Cleaving: Insulation of the reaction mixture in a
temperature at least 5.degree. C. above the melting temperature of
the target nucleic acid, and within the optimal operating
temperature of the restriction enzymes the said. The temperature
ranges from 45.about.89.degree. C., duration ranges from
1.about.300 seconds;
[0087] The temperatures of (1) denaturing, (3) elongation and (4)
cleaving steps are at least 10.degree. C. higher than the annealing
temperature in step (2). By repeated steps (1) to (4), say
denaturation, annealing, extension and cleaving, the target nucleic
acid molecules are amplified exponentially, the products include
double-stranded repetitive nucleic acid XRX/X'R'X', double-stranded
target nucleic acid X/X' and single-stranded target molecule X.
[0088] In practice, if the temperature of cleaving is the same with
that of extension, then step (4) and (3) can be combined into one
single step: Step (3) extension and cleaving, and the duration
ranges between 1-300 sec.
[0089] The schematic diagram of the mechanism of PECR amplification
reactions is shown in FIG. 1: Sense and antisense strands are
represented by solid and dashed lines respectively, the 3'-ends are
indicated by arrows and the restriction sites for PspGI are
indicated by solid diamonds. When a target oligonucleotide (X)
binds to a probe in the upstream, it is elongated by the Taq DNA
polymerase, and a full-duplex oligonucleotide containing tandem
repeats is produced. If the repeats are cleaved by PspGI, short
duplex target oligonucleotides (MC) are released; and when they are
not cleaved, the number of tandem repeats increases by slipping and
elongation. As shown in FIG. 1, PEAR consists of repetitive cycles
of: (1) heat denaturation, (2) annealing, (3) elongation, and (4)
cleaving. In the first cycle, a target oligonucleotide and an
antisense probe were heat-denatured and annealed to form a partial
duplex (X/X'R'X'). In the presence of dNTPs, they are elongated by
Taq DNA polymerase to form fully matched duplex tandem repeats
(XRX/X'R'X'). Subsequently, PspGI cleavage of the recognition site
releases monomeric oligonucleotides (X/X'). Thereafter, a next
cycle of denaturation, annealing, elongation and cleaving is
started again, resulting in exponential amplification of the duplex
oligonucleotide, and the amplification product is the
double-stranded target molecule X/X'.
[0090] In the step (2), when a target oligonucleotide binds to a
probe in the upstream (FIG. 1, top right), there is no elongation,
because it provides no primer/template structure for the Taq DNA
polymerase. This would affect the kinetics of PECR, but would not
lead to the stop of the PECR amplification because of the following
two reasons: (1) Both target and probe are present in a large
number of copies, according to the law of probability, nearly half
of the target oligonucleotides bind to the probe in the downstream,
and start the reaction; (2) Even if only one target molecule is
presented in the reaction, with several cycles of denaturation and
annealing, it will eventually bind to the probe's in the 3'-end,
and start the reaction.
[0091] In addition, in step (3) and (4), in the subsequent thermal
cycles, if the tandem repeated duplexes are not fully digested by
PspGI, because the duration of cleavage is rather short. When the
remaining tandem repeated duplexes are subjected to more cycles of
denaturing, reannealing and elongation, the number of repeat unit
increases continuously through slipped strand pairing and DNA
polymerase elongation (FIG. 1, bottom right). The slipping reaction
is linear, which can have an impact on the kinetics and the rate of
amplification of PECR reaction. However, provided sufficient amount
of restriction enzymes, most of the duplex repeats will be cut, so
it will not affect the exponential feature of the PECR
reaction.
[0092] In fact, when PspGI cleavage monomerizes the elongated
tandem repeats in a following cycle, many more duplex
oligonucleotides are released. It is this slipping-and-cleaving
mechanism that promotes not only the rate of amplification, but
also the yield of product. To facilitate the analysis and
detection, or applying in a subsequent reaction, if necessary, the
PEAR product is finally cleaved by PspGI for 10.about.60 minutes
after the completion of the thermal cycles, monomerizes the tandem
repeats fully into duplex oligonucleotides.
[0093] PECR product can be detected by the polyacrylamide gel
electrophoresis (PAGE). Preparing a non-denaturing polyacrylamide
gel with a concentration of 12%.about.15%, and 5.about.15 cm in
length, running through a 250.about.300V electrophoresis for
20.about.40 min, the DNA bands can be visualized by one of the
following methods:
[0094] (1) Staining the gel with ethidium bromide dye, then observe
and photograph the DNA bands with UV gel imaging system;
[0095] (2) Staining the gel with Sybr Green I or Sybr Green II dye,
and then observe and photograph the DNA bands with UV gel imaging
system;
[0096] (3) Reveal DNA bands by silver staining;
[0097] (4) Mix radioisotope labeled single deoxyribonucleotide into
the PECR reaction system, then operate electrophoresis followed by
autoradiography imaging.
[0098] Real-time fluorescence quantitative detection can also be
performed on PECR product with the following two methods:
[0099] (1) Adding fluorescent dye directly into the reaction
mixture:
[0100] Adding Sybr Green I or II fluorescent dye into the reaction,
Sybr Green binds specifically with the minor groove of DNA with
high affinity for double-stranded DNA (dsDNA), while its binding
capacity with single-stranded DNA (ssDNA) is very low. At the
beginning of PECR reaction, the probe is single-stranded, thus
binds with the Sybr Green weakly, and the fluorescence intensity is
at a relatively low level. During PECR reaction cycles,
single-stranded probe were converted into double-stranded products.
The fluorescence intensity enhance greatly due to Sybr Green dyes
bind with double-stranded products, which can be detected with a
fluorescence quantitative real-time PCR instrument, such as ABI
7500.
[0101] (2) Labeling PECR probe with fluorophore and quencher
groups:
[0102] Since Sybr Green dyes binds with dsDNA nonspecifically,
quantification of nucleic acids based on them have the
false-positive problem: if a false-positive or a nonspecific
amplification occurred, it is not distinguishable from a true
positive reaction. For the purpose of more accurate quantitative
detection, one can label the PECR probe with fluorophore and
quencher.
[0103] Fluorophores that can be used include but are not limited
to: 6-carboxyfluorescein (FAM), Tetrachlorofluorescein (TET),
hexachlorofluorescein (HEX),
N,N,N;N'-tetramethyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine(ROX),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), fluorescein
isothiocyanate (FITC),
3-(-carboxy-pentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine (CyA);
Texas Red, 6-carboxyrhodamine (R6G) etc. Quenchers include but not
limited to TARMA, Iowa Black (IWB), etc.
[0104] The principle of fluorescent labeling is shown in FIG. 2, in
which the fluorophore lies in the 5'-end of the probe, while the
quencher locates in the middle of which, more precisely, 3-10 bases
downstream the restriction enzyme cleavage site R'. At the
beginning of the PECR reaction, the fluorescence is close to the
quencher, according to the fluorescence resonance energy transfer
(FRET) principle most of the energy absorbed by the fluorophore
transfers to the quencher and releases as heat, therefore
fluorescence occurred in a lower level. Note that if the quencher
was set on the 3'-end of the probe, they would be too far apart
from each other, which may cause the quenching to be ineffective.
As PECR reaction occurs, single-stranded probes are continually
being converted into double-stranded products and being cut by
restriction enzymes, leading to the separation of the fluorophore
and the quencher. Energy absorbed by fluorophore will be released
in the form of fluorescence, with its signal being greatly
enhanced, which can therefore be detected by the fluorescence
quantitative real-time PCR instrument.
[0105] Specifically, all the dNTPs, DyNAzyme II Hot Start DNA
polymerase, restriction enzymes PspGI and buffer solution used in
this procedure are purchased from New England Biolabs, Co. Ltd,
Beijing branch. The synthetic oligonucleotides and probes are
purchased from Invitrogen Co. Ltd, Shanghai branch. The target
oligonucleotides (X) is derived from a human microRNA, hsa-miR-375,
its sequence is: 5'-TTTGTTCGTTCGGCTCGCGTGA-3'. In order to fasten
the rate of amplification, the probe (X'R'X'R'X') we adopted
contains 3 copies of the complementary sequence of hsa-miR-375,
which is:
TABLE-US-00001 5'-TCACGCGAGCCGAACGAACAAA-CCAGG-TCACGCGAGCCGAACGA
ACAAA-CCAGG-TCACGCGAGCCGAACGAACAAA-3'
[0106] The underlined shows the enzyme PspGI recognition and
cleaving sites.
[0107] In a 20 .mu.L volume reaction mixture, add 100-200 nM of
probe (X'R'X'R'X'), 10.sup.-1 to 10.sup.-12 uM of target
oligonucleotides (X), 0.02-0.1 Unit/uL of DyNAzyme II Hot Start DNA
Polymerase, 0.01-0.5 Unit/.mu.L of restriction enzyme PspGI,
1.times. of DyNAzyme II Hot Start DNA polymerase buffer and 50 uM
each dNTPs. The reactions were initiated at 90-95.degree. C. for
1-10 min for hot start and activation of the DNA polymerase
DyNAzyme II, followed by 20-40 cycles of denaturing at
90-95.degree. C. for 5-30 sec, annealing at 45-65.degree. C. for
5-30 sec, elongation and cleaving at 75.degree. C. for 1-5 min. If
necessary, PspGI digestion of the product is conducted by a final
incubation at 75.degree. C. for 10-60 min. PEAR products were
separated by 15% non-denaturing polyacrylamide gel electrophoresis
(PAGE), and visualized under an ultraviolet illuminator after SYBR
Gold staining, which is purchased from Molecular Probes.
[0108] To validate the reaction mechanism, PEAR reactions with
complete and incomplete (lacking Taq DNA polymerase, PspGI or
target) components were conducted under previously optimized
reaction conditions with target concentration at 1 nM and probe
concentration at 100 nM. As indicated by the arrow in FIG. 3, a
lower band (22 bp), representing the duplex monomers, X/X', and
several upper bands, representing tandem repeats, are observed in
the complete PEAR reactions. In addition, a higher concentration of
restriction enzyme (H in FIG. 3) produces a stronger band than a
low concentration of enzyme (L in FIG. 3). Such bands are not
observed if any of the four essential components (the two enzymes,
the target and the probe) is omitted. It is clear that the PECR
amplification depends on both of the two enzymes, the probe and the
target nucleic acid. FIG. 4 shows that PECR amplification is highly
sensitive and can detect target oligonucleotide as low as
10.sup.-10 .mu.M.
Embodiment 2
[0109] This embodiment is an example of reverse transcriptase PECR
(RT-PECR). RNA molecules, particularly small RNAs such as miRNA or
siRNA, were amplified by RT-PECR. Take miRNA as an example, the
reaction principle is shown in FIG. 5. The method comprises the
following steps:
[0110] Add poly-A tail to total miRNA using poly-A polymerase
(PAP);
[0111] Reverse transcript total miRNA to cDNA using Oligo-dT and
reverse transcriptase;
[0112] Remove RNA molecules from the product cDNA using RNase
H;
[0113] Amplify the target cDNA using PECR, the components of the
reaction mixtures and the thermal cycling parameters are the same
as those in embodiment 1.
Embodiment 3
[0114] This embodiment is RNA-direct PECR (RD-PECR), i.e., directly
amplify RNA molecules by PECR without reverse transcription. Take
miRNA as an example, the reaction principle is shown in FIG. 6, and
the method comprises the following four steps:
[0115] (1) Mix the PECR probe with total RNA directly, by heat
denaturation and annealing, the target miRNA bind to PECR probe to
form partial duplex miRNA/DNA hybrid molecules;
[0116] (2) Adding in the reaction mixture a DNA polymerase which
can directly extend RNA molecules, e.g., E. coli DNA polymerase I,
setting the temperature of the first thermal cycle to be the
optimum working temperature for the DNA polymerase I (37.degree.
C.), miRNA strands in the partially duplexes are extended at their
3'-end, forming target cDNA molecules whose sequences are the same
to the target miRNA;
[0117] (3) Remove RNA molecules from the product cDNA using RNase
H;
[0118] (4) Amplify the target cDNA using PECR, the components of
the reaction mixtures and the thermal cycling parameters are the
same as those in embodiment 1.
Embodiment 4
[0119] Choose 4 zebrafish miRNA as target, respectively miR-375,
miR-430a, miR-206 and miR-124. The sequence and function of miR-375
and miR-430a are known, they are selected for technical
verification. MiR-375 is necessary for pancreatic development,
reducing the level of miR-375 can inhibit the aggregation of islet
cells. The function of miR-430 is to clear maternal mRNAs that are
no longer needed in zebrafish embryos. In addition, it has been
reported that peaks of miR-430a, miR-206 and miR-124 expression
appeared respectively at 4 h, 12 h and 24 h after fertilization in
zebrafish embryos. This Embodiment performs a comparison of RT-PCR
and RT-PECR through analysis of miRNA expression in zebrafish early
embryo development.
[0120] (1) Total RNA Extraction and Reverse Transcription
[0121] Using Applied Biosystems mirVana miRNA Isolation Kit (Cat
#AM1560), total miRNA of zebrafish early embryos at 1 h, 2 h, 4 h,
12 h and 24 h after fertilized was extracted. Using Applied
Biosystems TaqMan miRNA Reverse Transcription Kit (Cat # 4366596),
the total miRNA was reverse transcript into cDNA, and used as
template in subsequent PCR and PECR reactions.
[0122] (2) Real-Time Quantitative PCR
[0123] Using glycerol 3-phosphate dehydrogenase (GAPDH) gene as
internal control, using Applied Biosystems TaqMan MicroRNA Assay
(Cat #4383443) and TaqMan Universal PCR Master Mix (Cat #4364338)
for quantitative detection of the target miRNA in the reverse
transcription products, the samples were standardized, and used as
external controls for PECR reactions.
[0124] (3) Real-Time Quantitative PECR
[0125] Using real-time quantitative PECR for quantitative detection
of the target miRNA in the samples, the components of the reaction
mixture and thermal cycling parameters are the same as those in
embodiment 1, except that the probes are labeled with a fluorophore
and a quencher. All reactions include a no-template control (NTC),
and were repeated three times at least. The reactions were
conducted in the Applied to Biosystems 7500 Real-Time PCR system,
and the fluorescence intensities were monitored in real-time as
PECR cycle changes.
[0126] (4) Results and Analysis
[0127] As shown in FIG. 7, real-time fluorescence detection
confirmed that fluorescence intensities increased with each PECR
cycle. Comparing the results of RT-PCR and RT-PECR, it was shown
that they have basically consistent results, indicated the high
accuracy of real-time quantitative PECR.
[0128] A universal primer must be used to match a miRNA-specific
primer when amplify miRNA by RT-PCR. It will possibly cause
non-specific amplification, false positives and false negatives
issues. However, when the detection is done by RT-PECR, only a
repeat-containing probe that is complementary to the target miRNA
is needed. Therefore, the PECR technology has characteristics of
simple, efficient and stable, and with higher specificity. So PECR
is potentially useful in amplification and quantitative analysis of
miRNA.
[0129] Throughout in this description and in the claims, the
singular may include the plural unless clearly stated. For example,
adding "a thermostable enzyme" in the reaction system includes
adding one or more kinds of thermostable enzymes; adding "a
thermostable DNA polymerase" in the reaction system concludes
adding one or more kinds of thermostable DNA polymerases; "a target
molecule" includes one or more target molecules; "a probe" includes
one or more probes, and so on.
[0130] In addition, this invention is not limited to the particular
configuration of the description. The terminology in the
description and the claims are only used for the description of a
specific implementation, but not to limit the invention to the
qualified range of terminology used, because the scope of the
invention is restricted only by the claims of rights and
requirements or articles which are equal to them.
[0131] One skilled in the art will understand that the embodiment
of the present invention as shown in the drawings and described
above is exemplary only and not intended to be limiting.
[0132] It will thus be seen that the objects of the present
invention have been fully and effectively accomplished. It
embodiments have been shown and described for the purposes of
illustrating the functional and structural principles of the
present invention and is subject to change without departure from
such principles. Therefore, this invention includes all
modifications encompassed within the spirit and scope of the
following claims.
* * * * *